Selective BRAFV600E Inhibitor PLX4720, Requires TRAIL Assistance to Overcome Oncogenic PIK3CA Resistance

Documented sensitivity of melanoma cells to PLX4720, a selective BRAFV600E inhibitor, is based on the presence of mutant BRAFV600E alone, while wt-BRAF or mutated KRAS result in cell proliferation. In colon cancer appearance of oncogenic alterations is complex , since BRAF, like KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. When PLX4720 was used to treat colon tumours, results were not encouraging and herein we attempt to understand the cause of this recorded resistance and discover rational therapeutic combinations to resensitize oncogene driven tumours to apoptosis. Treatment of two genetically different BRAFV600E mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKOBRAFV600E/PIK3CAH1047 cells. In contrast, for the same level of apoptosis in HT29BRAFV600E/PIK3CAP449T cells, TRAIL was combined with 17-AAG, an Hsp90 inhibitor. For cells where PLX4720 was completely ineffective, 17-AAG was alternatively used to target mutant BRAFV600E. TRAIL dependence on the constitutive activation of BRAFV600E is emphasised through the overexpression of BRAFV600E in the permissive genetic background of colon adenocarcinoma Caco-2 cells. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CAMT as the inhibitory factor. Another rational combination includes 17-AAG synergism with TRAIL in a BRAFV600E mutant dependent manner to commit cells to apoptosis, through DR5 and the amplification of the apoptotic pathway. We have successfully utilised combinations of two chemically unrelated BRAFV600E inhibitors in combination with TRAIL in a BRAFV600E mutated background and provided insight for new anti-cancer strategies where the activated PI3KCA mutation oncogene should be suppressed

Molecular biology and biochemistry of brain tumours

Elucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the b-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Molecular biology and biochemistry of brain tumours

ßElucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the ß-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Primer induced restriction analysis (PIRA) of a rarer and alternative polymorphism in the GHRH-R receptor, in which Thr replaces Ala at codon 57, in human GH-secreting pituitary tumours was investigated. Whilst the rarer form correlated with an increased response of the pituitary cells to GHRH in vitro, allele distribution studies revealed that it is unlikely that the polymorphism contributes to increased risk of developing GH-secreting tumours and therefore acromegaly. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type. Since this gene product is involved with development, these results suggest that p-catenin mutations may contribute to the initiation and subsequent growth of congenital adamantinomatous craniopharyngiomas

Μοριακή βιολογία και βιοχημεία των όγκων του εγκεφάλου

Elucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the β-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine / paracrine role of CCK in the control of growth and / or functioning of gliomas, meningiomas and pituitary tumours. Primer induced restriction analysis (PIRA) of a rarer and alternative polymorphism in the GHRH-R receptor, in which Thr replaces Ala at codon 57, in human GH-secreting pituitary tumours was investigated. Whilst the rarer form correlated with an increased response of the pituitary cells to GHRH in vitro, allele distribution studies revealed that it is unlikely that the polymorphism contributes to increased risk of developing GH-secreting tumours and therefore acromegaly. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between β-catenin gene alterations and craniopharyngiomas of the adamantinomatous type. Since this gene product is involved with development, these results suggest that β-catenin mutations may contribute to the initiation and subsequent growth of congenital adamantinomatous craniopharyngiomas.Η αποσαφήνιση ορισμένων μοριακών μηχανισμών και της βιοχημείας των όγκων του εγκεφάλου είναι ένα σημαντικό βήμα προς την ανάπτυξη επικουρικών ιατρικών θεραπειών. Η παρούσα μελέτη επικεντρώνεται στη χολοκυστοκίνη (CCK), ένα πεπτίδιο που απαντάται τόσο στο γαστρεντερικό όσο και στο εγκεφαλικό σύστημα, και έχει περιγραφεί ότι μπορεί να προκαλέσει μίτωση γλοιώματος αρουραίου καθώς και έκκριση ορμονών από την πρόσθια υπόφυση, μέσω του υποδοχέα CCK-B. Προσδιορίστηκε επίσης η σημασία ενός πολυμορφισμού στο γονίδιο υποδοχέα ορμόνης απελευθέρωσης αυξητικής ορμόνης (GHRH-R) (GHRH-R). Τέλος, διερευνήθηκαν ελαττώματα στο γονίδιο β-κατενίνης, ένα σημαντικό συστατικό της αναπτυξιακής οδού, σε ένα υποσύνολο κρανιοφαρυγγιομάτων. Αντίστροφη μεταγραφή-αλυσιδωτή αντίδραση πολυμεράσης (RT-PCR), ανάλυση περιοριστικής πέψης και άμεση αλληλούχιση έδειξε έκφραση του ίδιου του πεπτιδίου CCK και των υποδοχέων Α και Β από ανθρώπινα γλοιώματα, μηνιγγιώματα και όγκους της υπόφυσης. Τα πεπτίδια CCK διεγείρουν την ανάπτυξη καλλιεργημένων γλοιωμάτων και μηνιγγιώματος καθώς επίσης και την έκκριση in vitro ορμόνης [αυξητική ορμόνη (GH), ωχρινοτρόπου ορμόνη (LH) και ωοθυλακιοτρόπου ορμόνη (FSH)] από όγκους ανθρώπινης υπόφυσης. Αυτά τα βιολογικά αποτελέσματα μειώθηκαν ή αναστάλθηκαν από ανταγωνιστές CCK. Επιπλέον, ένα αντίσωμα ενάντια της CCK μείωσε τη μίτωση από γλοιώματα και μηνιγγιώματα, και το ίδιο αντίσωμα ανέστειλε την έκκριση ορμονών από καλλιεργημένους όγκους ανθρώπινης υπόφυσης. Τα πεπτίδια CCK διέγειραν την υδρόλυση φωσφατιδυλινοσιτόλης (ΡΙ), υποδεικνύοντας ότι η σύζευξη των υποδοχέων CCK με τη φωσπολιπάση C. Η κυκλική ΑΜΡ δεν επηρεάστηκε. Επιπλέον, η δραστικότητα κασπάσης-3 αυξήθηκε σημαντικά και σημαντικά, ενώ η δραστικότητα πρωτεασώματος μειώθηκε. Συνολικά, αυτά τα αποτελέσματα μπορεί να υποδηλώνουν έναν αυτόκρινο / παρακρινικό ρόλο του CCK στον έλεγχο της ανάπτυξης και / ή της λειτουργίας των γλοιωμάτων, μηνιγγιωμάτων και όγκων της υπόφυσης. Διεξήχθη ανάλυση περιορισμού προκαλούμενη από εκκινητή (PIRA) του σπανιότερου και εναλλακτικού πολυμορφισμού στον υποδοχέα GHRH-R, στον οποίο Thr αντικαθιστά το Ala στο κωδικόνιο 57, σε όγκους υπόφυσης που εκκρίνουν ανθρώπινη GH. Ενώ η σπανιότερη μορφή συσχετίζεται με αυξημένη απόκριση των κυττάρων της υπόφυσης στην GHRH in vitro, μελέτες κατανομής αλληλομορφών αποκάλυψαν ότι είναι απίθανο ο πολυμορφισμός να συμβάλει στον αυξημένο κίνδυνο ανάπτυξης όγκων που εκκρίνουν GH και συνεπώς ακρομεγαλίας. Περαιτέρω ευρήματα αυτής της μελέτης, χρησιμοποιώντας PCR και άμεση αλληλούχιση, ήταν η επίδειξη συσχέτισης μεταξύ μεταβολών γονιδίων β-κατατενίνης και κρανιοφαρυγγιομάτων του αδαμαντιωματώδους τύπου. Δεδομένου ότι αυτό το γονιδιακό προϊόν εμπλέκεται με την ανάπτυξη, αυτά τα αποτελέσματα υποδηλώνουν ότι οι μεταλλάξεις β-κατενίνης μπορεί να συμβάλλουν στην έναρξη και στη συνέχεια ανάπτυξη συγγενών αδαμαντινοσωματικών κρανιοφαρυγγιομάτων

BRAFV600E Efficient Transformation and Induction of Microsatellite Instability Versus KRASG12V Induction of Senescence Markers in Human Colon Cancer Cells12

In colorectal cancer, BRAF and KRAS oncogenes are mutated in about 15% and 35% respectively at approximately the same stage of the adenoma-carcinoma sequence. Since these two mutations rarely coexist, further analysis to dissect their function of transformation in colon cancer is required. Caco-2 human colon adenocarcinoma cells were stably transfected with BRAFV600E (Caco-BR cells) or KRASG12V (Caco-K cells) oncogenes. BRAFV600E is more efficient in transforming Caco-2 cells and altering their morphology. The dominant nature of BRAFV600E is evident by its ability to render Caco-2 cells tumorigenic in vivo all be it through selective extracellular signal-related kinase (ERK) 2 phosphorylation and high levels of cyclin D1. As a consequence, the cell cycle distribution of parental cells is altered and microsatellite instability is introduced. Attenuated ERK activation observed correlated with KSR downregulation by BRAFV600E without further implications to signaling. Highly activated ERK in case of KRASG12V (Caco-K cells) leads to mild transformation causing Caco-K cells to express premature senescence-related markers and acquire growth factor-dependent viability. Interestingly, BRAFWT gets equally activated by upstream KRAS mutations present in colon adenocarcinoma cells such as DLD-1 and SW620. Taken together, these results suggest that the two oncogenes have different transforming capability in colon cancer, although they both use the mitogen-activated protein (MAP) kinase pathway to carry out their effect. In general, BRAFV600E presents greater potential in mediating tumorigenic effect as compared to KRASG12V both in vivo and in vitro. These findings may have implications in personalised diagnosis and targeted therapeutics